JP7507785B2 - Method for extracting Yakuchi essential oil that inhibits Listeria monocytogenes and Yakuchi essential oil - Google Patents
Method for extracting Yakuchi essential oil that inhibits Listeria monocytogenes and Yakuchi essential oil Download PDFInfo
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- 239000000341 volatile oil Substances 0.000 title claims description 77
- 238000000034 method Methods 0.000 title claims description 18
- 241000186779 Listeria monocytogenes Species 0.000 title claims description 15
- 238000000605 extraction Methods 0.000 claims description 41
- 239000000843 powder Substances 0.000 claims description 33
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 30
- 235000013399 edible fruits Nutrition 0.000 claims description 18
- 239000007788 liquid Substances 0.000 claims description 10
- 238000001256 steam distillation Methods 0.000 claims description 6
- 238000000227 grinding Methods 0.000 claims description 5
- 238000007873 sieving Methods 0.000 claims description 4
- 238000007654 immersion Methods 0.000 claims 1
- 238000012360 testing method Methods 0.000 description 15
- 238000004821 distillation Methods 0.000 description 12
- 239000012153 distilled water Substances 0.000 description 11
- 238000002791 soaking Methods 0.000 description 11
- 230000002401 inhibitory effect Effects 0.000 description 7
- 230000005764 inhibitory process Effects 0.000 description 7
- 241000572565 Alpinia oxyphylla Species 0.000 description 6
- 241000894006 Bacteria Species 0.000 description 6
- 241000196324 Embryophyta Species 0.000 description 6
- 230000001580 bacterial effect Effects 0.000 description 6
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- 238000000691 measurement method Methods 0.000 description 5
- 230000035945 sensitivity Effects 0.000 description 5
- 239000000243 solution Substances 0.000 description 5
- 230000000844 anti-bacterial effect Effects 0.000 description 4
- 238000002386 leaching Methods 0.000 description 4
- 239000002609 medium Substances 0.000 description 4
- 239000002245 particle Substances 0.000 description 4
- GSDSWSVVBLHKDQ-JTQLQIEISA-N Levofloxacin Chemical compound C([C@@H](N1C2=C(C(C(C(O)=O)=C1)=O)C=C1F)C)OC2=C1N1CCN(C)CC1 GSDSWSVVBLHKDQ-JTQLQIEISA-N 0.000 description 3
- 230000003385 bacteriostatic effect Effects 0.000 description 3
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- C—CHEMISTRY; METALLURGY
- C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
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- C11B9/00—Essential oils; Perfumes
- C11B9/02—Recovery or refining of essential oils from raw materials
- C11B9/027—Recovery of volatiles by distillation or stripping
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N65/00—Biocides, pest repellants or attractants, or plant growth regulators containing material from algae, lichens, bryophyta, multi-cellular fungi or plants, or extracts thereof
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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- A01N65/00—Biocides, pest repellants or attractants, or plant growth regulators containing material from algae, lichens, bryophyta, multi-cellular fungi or plants, or extracts thereof
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- A01N65/48—Zingiberaceae [Ginger family], e.g. ginger or galangal
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- C—CHEMISTRY; METALLURGY
- C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
- C11B—PRODUCING, e.g. BY PRESSING RAW MATERIALS OR BY EXTRACTION FROM WASTE MATERIALS, REFINING OR PRESERVING FATS, FATTY SUBSTANCES, e.g. LANOLIN, FATTY OILS OR WAXES; ESSENTIAL OILS; PERFUMES
- C11B9/00—Essential oils; Perfumes
- C11B9/02—Recovery or refining of essential oils from raw materials
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
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Description
本発明は、生物医薬の技術分野に属し、具体的にはリステリア・モノサイトゲネスを阻害するヤクチ精油の抽出方法及びヤクチ精油に関する。 The present invention belongs to the technical field of biopharmaceuticals, and specifically relates to a method for extracting Yakuchi essential oil that inhibits Listeria monocytogenes, and to Yakuchi essential oil.
ヤクチ(ラテン名:Alpinia oxyphylla Miq)は、別名がヤクチニン、ヤクチシであり、アルピニア種多年生草本植物である。ヤクチは、中国の4つの主な南部の薬物の1つであり、生薬として使用できるだけでなく、広範な薬理作用も持つ。雲ケイ瑩ら(ヤクチニン精油の酸化防止及び抗菌能力についての分析及び研究[J].弘光学報、2013、(第71期))は、超臨界CO2抽出法でヤクチ精油を抽出し、その酸化防止及び抗菌能力を分析し、結果として、抽出されたヤクチ精油が大腸菌及び黄色ブドウ球菌に対して顕著な阻害効果を有することを発見した。羅琴ら(水蒸気蒸留法によるヤクチニン揮発性油の抽出プロセスの最適化及びその体外静菌活性についての研究[J].華西薬学雑誌、2011、26(02):147-149)は、水蒸気蒸留法でヤクチニン揮発性油を抽出し、結果として、ヤクチニン揮発性油が大腸菌、黄色ブドウ球菌及び緑膿菌に対していずれも明らかな阻害作用を有することを発見した。現在、ヤクチ精油がリステリア・モノサイトゲネスに対して阻害作用を有することに関する報告はない。 Alpinia oxyphylla Miq (Latin name: Alpinia oxyphylla Miq), also known as Alpinia oxyphylla and Alpinia oxyphylla, is a perennial herbaceous plant of the Alpinia species. Alpinia oxyphylla Miq is one of the four main southern Chinese medicines, which can not only be used as a crude drug, but also has a wide range of pharmacological effects. Yun Jingying et al. (Analysis and Research on the Antioxidant and Antibacterial Ability of Alpinia oxyphylla Essential Oil [J]. Hongguang Report, 2013, (71st issue)) extracted Alpinia oxyphylla essential oil by supercritical CO2 extraction method, analyzed its antioxidant and antibacterial ability, and found that the extracted Alpinia oxyphylla essential oil has a significant inhibitory effect on Escherichia coli and Staphylococcus aureus. Luo Qin et al. (Research on the optimization of the extraction process of yakuchinin volatile oil by steam distillation and its in vitro bacteriostatic activity [J]. West China Pharmaceutical Journal, 2011, 26(02):147-149) extracted yakuchinin volatile oil by steam distillation and found that yakuchinin volatile oil had obvious inhibitory effects on Escherichia coli, Staphylococcus aureus and Pseudomonas aeruginosa. Currently, there are no reports on the inhibitory effect of yakuchi essential oil on Listeria monocytogenes.
リステリア・モノサイトゲネス(Listeria monocytogenes)は、人獣共通感染症の病原体であり、自然界に広く存在しており、食品由来のリステリア・モノサイトゲネスは、人間の安全に危険をもたらし、該細菌は、4℃の環境でも成長し繁殖することができ、冷蔵食品が人間の健康を脅かす主な病原菌の1つである。該細菌は、物理化学的因子に対して高い耐性があり、土壌、糞便、サイレージなどの様々な環境でいずれも長期間生存することができ、アルカリ及び塩に対して高い耐性があり、凍結及び解凍するのが困難であり、高い浸透圧に耐えることができ、抑制したり殺したりするのが困難である。出願人は、研究によると、特定の方法で抽出したヤクチ精油がリステリア・モノサイトゲネスに対して明らかな阻害作用を示すことを発見した。 Listeria monocytogenes is a zoonotic pathogen that is widely present in nature, and food-borne Listeria monocytogenes poses a danger to human safety. The bacterium can grow and reproduce even at 4°C, and is one of the main pathogens that refrigerated foods pose to human health. The bacterium has high resistance to physicochemical factors, can survive for a long time in various environments such as soil, feces, and silage, has high resistance to alkali and salt, is difficult to freeze and thaw, can withstand high osmotic pressure, and is difficult to inhibit or kill. The applicant has found through research that Yakchi essential oil extracted by a specific method has obvious inhibitory effect on Listeria monocytogenes.
精油を異なる抽出方法で抽出する場合にその化学成分なども大きく異なり、精油の用途に応じて適切な抽出方法を選択する必要がある(王瑶ら、2017、Bakkali et al、2008)。したがって、本発明がリステリア・モノサイトゲネスを阻害するヤクチ精油の抽出方法を開示することは、ヤクチ精油の普及及び応用に対して重要な意味を有する。 When essential oils are extracted using different extraction methods, their chemical components vary greatly, and it is necessary to select an appropriate extraction method depending on the intended use of the essential oil (Wang Yao et al., 2017; Bakkali et al., 2008). Therefore, the disclosure of the extraction method for Yakuchi essential oil that inhibits Listeria monocytogenes by the present invention is of great significance for the popularization and application of Yakuchi essential oil.
従来技術の欠点に鑑み、上記問題を解決するために、本発明の目的は、リステリア・モノサイトゲネスを阻害するヤクチ精油の抽出方法及びヤクチ精油を提供することである。 In view of the shortcomings of the prior art and in order to solve the above problems, the object of the present invention is to provide a method for extracting Yakuchi essential oil that inhibits Listeria monocytogenes, and Yakuchi essential oil.
本発明の解決手段は、以下の内容を含む。 The solution of the present invention includes the following:
リステリア・モノサイトゲネスを阻害するヤクチ精油の抽出方法は、
ヤクチの乾燥した成熟果実を粉砕して篩にかけて、粉末を得ることと、粉末と水とを固液質量比1:10~12で混合して2~3h静置して浸漬して、220~300℃で5~7h水蒸気蒸留することとを含む。
The method for extracting Yakuchi essential oil that inhibits Listeria monocytogenes is as follows:
The process involves crushing and sieving dried, mature fruits of the yakuchi to obtain a powder, mixing the powder with water in a solid-liquid mass ratio of 1:10-12, leaving the mixture to soak for 2-3 hours, and then steam distilling the mixture at 220-300°C for 5-7 hours.
好ましくは、粉砕して篩にかけることにより得られた粉末を、まず35±5℃で、相対湿度60~70%の条件下で静置し、次に水を加えて浸漬して水蒸気蒸留する。 Preferably, the powder obtained by crushing and sieving is first left to stand at 35±5°C and a relative humidity of 60-70%, and then water is added to soak the powder and steam distilled.
好ましくは、前記静置時間が5~7dである。 Preferably, the standing time is 5 to 7 days.
好ましくは、前記篩が40~60メッシュの篩である。 Preferably, the sieve is a 40 to 60 mesh sieve.
好ましくは、粉末と水とを混合する場合、水のpHが塩基性である。 Preferably, when the powder is mixed with water, the pH of the water is basic.
好ましくは、粉末と水とを混合する場合、水のpHが7.5~8.5である。 When mixing the powder with water, the pH of the water is preferably 7.5 to 8.5.
好ましくは、粉末と水とを混合する場合、水のpHが8.5である。 Preferably, when mixing the powder with water, the pH of the water is 8.5.
好ましくは、水蒸気蒸留の温度が220~230℃である。 Preferably, the temperature of the steam distillation is 220 to 230°C.
他の態様では、本発明は、ヤクチ精油を提供し、該精油は、本発明に係る方法で製造されたものであり、リステリア・モノサイトゲネスに対して阻害活性を示す。 In another aspect, the present invention provides a Yakuchi essential oil, the essential oil being produced by the method of the present invention and exhibiting inhibitory activity against Listeria monocytogenes.
本発明によって達成される有益な効果は以下のとおりである。 The beneficial effects achieved by the present invention are as follows:
本発明に係るリステリア・モノサイトゲネスを阻害するヤクチ精油の抽出方法は、水蒸気蒸留法で該精油の高効率抽出を実現し、抽出率が1.5%以上である。 The method for extracting Yakuchi essential oil that inhibits Listeria monocytogenes according to the present invention uses steam distillation to achieve highly efficient extraction of the essential oil, with an extraction rate of 1.5% or more.
本発明の抽出方法は、操作しやすく、化学試薬を大量に使用する必要がなく、安全かつ効率的である。 The extraction method of the present invention is easy to operate, does not require the use of large amounts of chemical reagents, and is safe and efficient.
本発明により得られたヤクチ精油は、リステリア・モノサイトゲネスに対して非常に高い阻害活性を示す。阻止円の直径は8.6mm以上であり、MICは15μL/mL以下である。 The Yakuchi essential oil obtained by the present invention exhibits extremely high inhibitory activity against Listeria monocytogenes. The inhibition zone diameter is 8.6 mm or more, and the MIC is 15 μL/mL or less.
本発明の技術内容をよりよく理解するために、具体的な実施例を以下に提供して、本発明をさらに説明する。 In order to better understand the technical content of the present invention, specific examples are provided below to further illustrate the present invention.
前記ヤクチは、ヤクチAlpinia oxyphylla Miq. の乾燥した成熟果実であり、サンプルが海南省白砂県擁処村で採取された。 The yakuchi is the dried mature fruit of the yakuchi Alpinia oxyphylla Miq., and the sample was collected from Baichuo Village, Baisha County, Hainan Province.
試験例1 粉砕度への考察
ヤクチの乾燥した成熟果実をそれぞれ粉砕して10、20、30、40、50、60メッシュの篩にかけて、粒子サイズの異なる粉末を得て、粒子サイズの異なる粉末をそれぞれ50g秤量し、10倍の質量のpH6.8の蒸留水を添加し、3h浸漬し、220℃で5h水蒸気蒸留し、試験を3回繰り返した。2020年版の《中国薬典》で規定された精油測定法の「方法I」に従って精油抽出率を測定した。精油抽出率(%)=精油体積(mL)/植物粉末質量(g)×100%。
Test Example 1 Consideration of the degree of crushing Dried mature fruits of Yakchi were crushed and sieved through 10, 20, 30, 40, 50, and 60 mesh sieves to obtain powders with different particle sizes. 50 g of each powder with different particle sizes was weighed, 10 times the mass of distilled water with a pH of 6.8 was added, soaked for 3 hours, and steam distilled at 220 ° C for 5 hours, and the test was repeated three times. The essential oil extraction rate was measured according to "Method I" of the essential oil measurement method specified in the 2020 edition of the Chinese Pharmacopoeia. Essential oil extraction rate (%) = essential oil volume (mL) / plant powder mass (g) x 100%.
結果を以下に示した:サンプル粒子が10メッシュから40メッシュに減少すると、ヤクチ精油の抽出率が急激に上昇し、拡散の法則によれば、ヤクチ果実の粉砕度が高いほど、蒸留水との接触が多いほど、浸出効果が高く、精油抽出率が高い。サンプル粒子が40メッシュから60メッシュに減少すると、ヤクチ精油の抽出率がほぼ安定するまで徐々に低下する。これは、ヤクチ果実の粉砕度が高すぎて、水蒸気蒸留中、ヤクチ果実から浸出された成分が粉砕度の向上に伴って増加し、水に浸漬された成分が多すぎると精油が水蒸気に伴って蒸発する抵抗を上げて、精油抽出率の低下をもたらすことによるものである可能性がある。したがって、粉砕度が40メッシュ(抽出率が1.7%である)を選択する。 The results are shown below: When the sample particles are reduced from 10 mesh to 40 mesh, the extraction rate of Yakchi essential oil increases sharply. According to the law of diffusion, the higher the degree of grinding of Yakchi fruit and the more contact with distilled water, the higher the leaching effect and the higher the essential oil extraction rate. When the sample particles are reduced from 40 mesh to 60 mesh, the extraction rate of Yakchi essential oil gradually decreases until it is almost stable. This may be due to the fact that the degree of grinding of Yakchi fruit is too high, and the components extracted from Yakchi fruit during steam distillation increase with the improvement of the degree of grinding, and too many components immersed in water increase the resistance of essential oil to evaporate with steam, resulting in a decrease in the essential oil extraction rate. Therefore, the degree of grinding is selected to be 40 mesh (the extraction rate is 1.7%).
試験例2 固液比への考察
ヤクチの乾燥した成熟果実を粉砕して40メッシュの篩にかけて、粉末を得て、粉末を50g秤量し、7、8、9、10、11、12倍の質量のpH6.8の蒸留水をそれぞれ添加し、3h浸漬し、220℃で5h水蒸気蒸留し、試験を3回繰り返した。2020年版の《中国薬典》で規定された精油測定法の「方法I」に従って精油抽出率を測定した。精油抽出率(%)=精油体積(mL)/植物粉末質量(g)×100%。
Test Example 2 Consideration of solid-liquid ratio Dried mature fruit of Yakchi was crushed and sieved through a 40 mesh sieve to obtain powder, and 50 g of the powder was weighed, and 7, 8, 9, 10, 11, and 12 times the mass of distilled water with a pH of 6.8 was added, soaked for 3 hours, and steam distilled at 220 ° C for 5 hours, and the test was repeated three times. The essential oil extraction rate was measured according to "Method I" of the essential oil measurement method specified in the 2020 edition of the Chinese Pharmacopoeia. Essential oil extraction rate (%) = essential oil volume (mL) / plant powder mass (g) x 100%.
結果を以下に示した:固液比が1:5~12である場合、抽出率は徐々に高くなり、固液比が1:10である場合、抽出率は最大1.7%である。 The results are shown below: When the solid-liquid ratio is 1:5-12, the extraction rate gradually increases, and when the solid-liquid ratio is 1:10, the extraction rate is up to 1.7%.
固液比が7~10倍である場合、ヤクチ精油の抽出率は急激に上昇する。固液比が10~12倍である場合、ヤクチ精油の抽出率はまず低下し、その後に変化せず(抽出率が1.5~1.7%である)、水量の増加に伴って、サンプルと蒸留水との接触面積が大きくなり、精油の浸出と水中への拡散を促進する。固液比が増加し続け、精油量が増加しない場合、過剰な水溶液により精油の蒸留水への溶解を増加させて、精油が水蒸気に伴って揮発しにくくなり、精油抽出率を低下させるため、固液比を10倍に選択する。 When the solid-liquid ratio is 7-10 times, the extraction rate of Yakchi essential oil increases sharply. When the solid-liquid ratio is 10-12 times, the extraction rate of Yakchi essential oil first decreases and then remains unchanged (extraction rate is 1.5-1.7%), and as the amount of water increases, the contact area between the sample and distilled water increases, promoting the leaching and diffusion of essential oil into the water. When the solid-liquid ratio continues to increase and the amount of essential oil does not increase, the excess aqueous solution increases the dissolution of essential oil in distilled water, making it difficult for essential oil to volatilize with water vapor, reducing the essential oil extraction rate, so the solid-liquid ratio is selected to be 10 times.
試験例3 浸漬時間への考察
ヤクチの乾燥した成熟果実を粉砕して40メッシュの篩にかけて、粉末を得て、粉末を50g秤量し、10倍量のpH6.8の蒸留水でそれぞれ0、1、2、3、4、5h浸漬し、220℃で5h水蒸気蒸留し、試験を3回繰り返した。2020年版の《中国薬典》で規定された精油測定法の「方法I」に従って精油抽出率を測定した。精油抽出率(%)=精油体積(mL)/植物粉末質量(g)×100%。
Test Example 3 Consideration of soaking time Dried mature fruit of Yakchi was crushed and sieved through a 40 mesh sieve to obtain powder, and 50 g of the powder was weighed out and soaked in 10 times the amount of distilled water with a pH of 6.8 for 0, 1, 2, 3, 4, and 5 hours, respectively, and steam distilled at 220 ° C for 5 hours, and the test was repeated three times. The essential oil extraction rate was measured according to "Method I" of the essential oil measurement method specified in the 2020 edition of the Chinese Pharmacopoeia. Essential oil extraction rate (%) = essential oil volume (mL) / plant powder mass (g) x 100%.
結果を以下に示した:浸漬時間が0~2hである場合、浸漬時間とヤクチ精油の抽出率が正の相関で増加する。これは、所定の時間範囲内で浸漬時間が水中への精油の分散効果を決定でき、浸漬時間が長いほど、水による分散作用が大きくなることによるものである可能性がある。浸漬時間が2~5hである場合、浸漬時間とヤクチ精油の抽出率が逆相関し、ヤクチ果実精油の抽出量の損失が非常に多い。これは、浸漬時間が長すぎて一部の精油成分が揮発して、精油の抽出率を大幅に低下させることによるものである可能性があり、したがって、浸漬時間を2~3hに選択する。 The results are shown below: When the soaking time is 0-2h, the soaking time and the extraction rate of Yakchi essential oil increase in a positive correlation. This may be because within a certain time range, the soaking time can determine the dispersion effect of essential oil in water, and the longer the soaking time, the greater the dispersion effect by water. When the soaking time is 2-5h, the soaking time and the extraction rate of Yakchi essential oil are inversely correlated, and the loss of the extraction amount of Yakchi fruit essential oil is very large. This may be because the soaking time is too long, causing some essential oil components to volatilize, which greatly reduces the extraction rate of essential oil, so the soaking time is selected to be 2-3h.
試験例4 蒸留時間への考察
ヤクチの乾燥した成熟果実を粉砕して40メッシュの篩にかけて、粉末を得て、粉末を50g秤量し、10倍量のpH6.8の蒸留水で2h浸漬し、220℃でそれぞれ2、3、4、5、6、7、8h水蒸気蒸留し、試験を3回繰り返した。2015年版の《中国薬典》で規定された精油測定法の「方法I」に従って精油抽出率を測定した。精油抽出率(%)=精油体積(mL)/植物粉末質量(g)×100%。
Test Example 4 Consideration of distillation time Dried mature fruit of Yakchi was crushed and sieved through a 40 mesh sieve to obtain powder, 50g of powder was weighed, soaked in 10 times the amount of distilled water with a pH of 6.8 for 2h, and steam distilled at 220°C for 2, 3, 4, 5, 6, 7, and 8h, respectively, and the test was repeated three times. The essential oil extraction rate was measured according to "Method I" of the essential oil measurement method specified in the 2015 edition of the Chinese Pharmacopoeia. Essential oil extraction rate (%) = essential oil volume (mL) / plant powder mass (g) x 100%.
結果を以下に示した:蒸留時間が2~5hである場合、ヤクチ精油の抽出率が急激に上昇し、精油の浸出量が蒸留時間に比例し、5hで最大値になって変化しない。これは、主に、蒸留時間が長くなるにつれて、精油が水蒸気に伴って揮発し続け、所定の時間になると、ヤクチ果実の精油成分全体が浸出し、浸出量が最高値になるからである。5h浸漬すると、抽出率は最大値(1.65%)なって変化しなかった。蒸留時間が5~8hである場合、抽出率は大きく変化しなかった。したがって、単変量試験によって、エネルギー及び時間を節約する視点から、蒸留時間を5hに選択する。 The results are shown below: When the distillation time is 2-5h, the extraction rate of yakuchi essential oil increases rapidly, and the amount of essential oil leaching is proportional to the distillation time, reaching a maximum value at 5h and remaining unchanged. This is mainly because as the distillation time increases, the essential oil continues to evaporate with the water vapor, and at a certain time, the entire essential oil components of the yakuchi fruit are leached out and the amount of leaching reaches its maximum value. After 5h of soaking, the extraction rate reaches a maximum value (1.65%) and remains unchanged. When the distillation time is 5-8h, the extraction rate does not change significantly. Therefore, through univariate testing, the distillation time is selected to be 5h from the perspective of saving energy and time.
試験例5 蒸留温度への考察
ヤクチの乾燥した成熟果実を粉砕して40メッシュの篩にかけて、粉末を得て、粉末を50g秤量し、10倍量のpH6.8の蒸留水で2h浸漬し、それぞれ100、140、180、220、260、300℃で5h水蒸気蒸留し、試験を3回繰り返した。2015年版の《中国薬典》で規定された精油測定法の「方法I」に従って精油抽出率を測定した。精油抽出率(%)=精油体積(mL)/植物粉末質量(g)×100%。
Test Example 5 Consideration of distillation temperature Dried mature fruit of Yakchi was crushed and sieved through a 40 mesh sieve to obtain powder, 50g of powder was weighed, soaked in 10 times the amount of distilled water with pH 6.8 for 2h, and steam distilled at 100, 140, 180, 220, 260, and 300°C for 5h, respectively, and the test was repeated three times. The essential oil extraction rate was measured according to "Method I" of the essential oil measurement method specified in the 2015 edition of the Chinese Pharmacopoeia. Essential oil extraction rate (%) = essential oil volume (mL) / plant powder mass (g) x 100%.
結果を以下に示した:蒸留温度が100~220℃である場合、抽出率が上昇する傾向にあり、蒸留温度が220~300℃である場合、抽出率が徐々に低下する。温度が上昇するにつれて、サンプル中の揮発性成分は蒸発し続け、蒸留温度が高すぎると、サンプル中の揮発性成分のごく一部は冷却し収集せずに空気へ蒸発するため、温度が高すぎると、精油抽出率は徐々に低下し、蒸留温度が220℃である場合、ヤクチ果実精油の抽出率は最も高く、1.75%である。 The results are shown below: when the distillation temperature is 100-220°C, the extraction rate tends to increase, and when the distillation temperature is 220-300°C, the extraction rate gradually decreases. As the temperature increases, the volatile components in the sample continue to evaporate, and when the distillation temperature is too high, a small part of the volatile components in the sample evaporates into the air without being cooled and collected, so when the temperature is too high, the essential oil extraction rate gradually decreases, and when the distillation temperature is 220°C, the extraction rate of yakuchi fruit essential oil is the highest, which is 1.75%.
単変量実験及びスターテストにより、最終的にヤクチ果実精油の条件として、粉砕度が40メッシュで、固液比が1:10で、230℃で2.4h浸漬し、5.5h蒸留することを決定し、抽出率が1.8%である。 Through univariate experiments and star tests, the final conditions for yakuchi fruit essential oil were determined to be a crushing degree of 40 mesh, a solid-liquid ratio of 1:10, soaking at 230°C for 2.4 hours, and distillation for 5.5 hours, with an extraction rate of 1.8%.
また、研究によると、一部のサンプルがリステリア・モノサイトゲネスに対してより高い静菌活性を示すことを発見した。比較試験の結果は以下のとおりである。 The study also found that some samples showed higher bacteriostatic activity against Listeria monocytogenes. The results of the comparative test are as follows:
試験例6 静菌活性への研究
6.1 材料、菌種、試薬
材料:精油1)~5):ヤクチの乾燥した成熟果実を粉砕して40メッシュの篩にかけて、粉末を得て、粉末を50g秤量し、それぞれ10倍量のpH7.0、7.5、8.0、8.5、9.0の蒸留水で2.4h浸漬し、230℃で5.5h水蒸気蒸留して得られたものである。精油6):ヤクチの乾燥した成熟果実を粉砕して40メッシュの篩にかけて、粉末を得て、粉末を50g秤量し、10倍量のpH7.0の蒸留水で2.4h浸漬し、180℃で5.5h水蒸気蒸留して得られたものである。精油7)~10):ヤクチの乾燥した成熟果実を粉砕して40メッシュの篩にかけて、粉末を得て、粉末を35±5℃で相対湿度65±5%で(厚さ2~3cmで)それぞれ3、5、7、9d静置し、粉末を50g秤量し、10倍量のpH8.5の蒸留水で2.4h浸漬し、180℃で5.5h水蒸気蒸留して得られたものである。
Test Example 6 Research on bacteriostatic activity 6.1 Materials, fungus species, reagents Materials: Essential oils 1) to 5): Dried mature fruit of Yakuchi was crushed and sieved through a 40-mesh sieve to obtain powder, 50 g of the powder was weighed, soaked in 10 times the amount of distilled water with pH 7.0, 7.5, 8.0, 8.5, and 9.0 for 2.4 hours, and steam distilled at 230 ° C for 5.5 hours to obtain. Essential oil 6): Dried mature fruit of Yakuchi was crushed and sieved through a 40-mesh sieve to obtain powder, 50 g of the powder was weighed, soaked in 10 times the amount of distilled water with pH 7.0 for 2.4 hours, and steam distilled at 180 ° C for 5.5 hours to obtain. Essential oils 7) to 10): Dried ripe fruits of Yakuchi were crushed and sieved through a 40-mesh sieve to obtain powder. The powder was allowed to stand at 35±5°C and 65±5% relative humidity (at a thickness of 2 to 3 cm) for 3, 5, 7, and 9 days, respectively. 50 g of the powder was weighed out, immersed in 10 times the amount of distilled water with a pH of 8.5 for 2.4 hours, and steam distilled at 180°C for 5.5 hours to obtain the essential oils.
菌種とリステリア・モノサイトゲネス(Listeria monocytogenes ATCC 19111)、及び黄色ブドウ球菌(Staphylococcus aureus ATCC 6538)は、いずれも北京北納創聯生物工学研究院から購入された。 The bacterial strains, Listeria monocytogenes (ATCC 19111), and Staphylococcus aureus (ATCC 6538) were all purchased from Beijing Beina Chuanglian Institute of Biotechnology.
試薬:培地、レボフロキサシンなどは、いずれも北京索ライ宝科学技術有限公司から購入された。 Reagents: Culture medium, levofloxacin, etc. were all purchased from Beijing Suo Laibao Science and Technology Co., Ltd.
6.2 試験方法
6.2.1 阻止円の測定
濾紙寒天ディスク拡散法を使用して、ピペットを用いて20μLの精油又はコントロールを滅菌環境条件下で滅菌濾紙(d=6mm)に滴下し、濾紙が精油又はコントロールを完全に吸収してから、各細菌溶液(細菌溶液の濃度が107cfu/mlで、塗布量が60μLである)を均一に塗布した培地プレート(TSA、d=6cm)に当該濾紙を貼り付け、マークを付け、各細菌に対して3回繰り返し、陰性コントロールが滅菌水であり、陽性コントロールがレボフロキサシン(濃度15μg/ml)であり、培養条件がインキュベータ内で37℃で24h培養することである。培養した後、阻止円の有無及び直径の大きさを感度の判断基準として観察して記録し、クロス法で阻止円の直径を測定し、その平均値を測定結果とした。結果標準は、阻止円の直径が20mmを超えると、感度が非常に高いとし、直径が15~20mmであると、感度が高いとし、直径が10~15mmであると、感度が中程度であるとし、直径が10mmよりも小さいと、感度が低いとする(蘭仕梅ら、2018)ことである。
6.2 Test Method 6.2.1 Measurement of Inhibition Circle Using the filter paper agar disk diffusion method, 20μL of essential oil or control is dropped onto a sterile filter paper (d=6mm) using a pipette under sterile environmental conditions, and after the filter paper completely absorbs the essential oil or control, the filter paper is attached to a medium plate (TSA, d=6cm) evenly coated with each bacterial solution (concentration of bacterial solution is 107 cfu/ml, application amount is 60μL), marked, and repeated three times for each bacterium, the negative control is sterile water, the positive control is levofloxacin (concentration 15μg/ml), and the incubation conditions are incubation in an incubator at 37℃ for 24h. After incubation, the presence or absence of inhibition circle and the size of its diameter are observed and recorded as the sensitivity judgment criterion, and the diameter of the inhibition circle is measured by the cross method, and the average value is the measurement result. The outcome standard is that a diameter of inhibition circle greater than 20 mm indicates very high sensitivity, a diameter of 15-20 mm indicates high sensitivity, a diameter of 10-15 mm indicates medium sensitivity, and a diameter smaller than 10 mm indicates low sensitivity (Lansimei et al., 2018).
6.2.2 最小発育阻止濃度(MIC)
MIC試験を微量二倍希釈法で96ウェルプレートで実施し、A1を第1のウェルとして使用して、左から右に順に行い、各プレートの1~12個のウェルに薬液を入れ、プレートごとに3つのブランク、陽性コントロール及び陰性コントロールを作成し、陰性コントロールが滅菌水であり、陽性コントロールがレボフロキサシンである。まずピペットを用いて各列の第1のウェルに180μLの各細菌懸濁液(細菌濃度107cfu/m、塗布量60μL)を添加し、第2~12のウェルにそれぞれ100μLの各細菌懸濁液を添加し、次に20μLの精油を第1のウェルにそれぞれ吸引し、精油の濃度をそれぞれ100、50、25、12.5、6.25、3.125、1.563、0.781、0.391、0.195、0.097μL/mLにするように第1~12のウェルに対して順に段階希釈を行い、十分かつ均一に混合し、3回繰り返し、培養条件がインキュベータ内で37℃で24h培養することであり、その後にマイクロプレートリーダーで真菌の吸光度を測定し、抗菌率が80%であるサンプルの濃度を該細菌に対する該サンプルの最小発育阻止濃度(MIC)とする。
6.2.2 Minimum inhibitory concentration (MIC)
The MIC test was performed in a 96-well plate by microdilution double dilution method, using A1 as the first well, from left to right, and drug solutions were added to 1-12 wells of each plate, and 3 blanks, positive control and negative control were prepared for each plate, the negative control was sterile water, and the positive control was levofloxacin. First, add 180 μL of each bacterial suspension (bacterial concentration 10 7 cfu/m, application amount 60 μL) to the first well of each row using a pipette, add 100 μL of each bacterial suspension to the second to twelfth wells, then aspirate 20 μL of essential oil into the first well, and serially dilute the essential oil in the first to twelfth wells in order to make the essential oil concentrations 100, 50, 25, 12.5, 6.25, 3.125, 1.563, 0.781, 0.391, 0.195, and 0.097 μL/mL, respectively, mix thoroughly and uniformly, and repeat three times. The culture conditions are to culture in an incubator at 37°C for 24 hours, and then measure the absorbance of the fungus with a microplate reader, and the concentration of the sample with an antibacterial rate of 80% is the minimum inhibitory concentration (MIC) of the sample against the bacterium.
6.2.3 最小殺菌濃度(MBC)の測定
寒天培地プレート法を使用して、「6.2.2」での各サンプルのMIC値に対応する溶液を対応する培地に均一に塗布し、培養条件がインキュベータ内で28℃で培養することであり、その後に培地において菌株が増殖しているか否かを観察し、該薬物のMBCはコロニー数が5個よりも少ないプレートに対応するウェルでの最低濃度である。
6.2.3 Determination of Minimum Bactericidal Concentration (MBC) Using the agar plate method, the solution corresponding to the MIC value of each sample in "6.2.2" is evenly spread on the corresponding medium, and the culture condition is to culture in an incubator at 28°C, and then observe whether the strain grows in the medium, and the MBC of the drug is the lowest concentration in the well corresponding to the plate with the number of colonies less than 5.
6.3 結果及び分析
結果を表1~2に示す。結果は、得られたヤクチ精油が、リステリア・モノサイトゲネスに対して優れた阻害作用を示し、殺菌活性がないことを示した。そのうち、精油8)と精油9)がより高い阻害活性を示した。
6.3 The results and analysis results are shown in Tables 1 and 2. The results showed that the obtained Yakuchi essential oils exhibited excellent inhibitory activity against Listeria monocytogenes and had no bactericidal activity. Among them, essential oil 8) and essential oil 9) showed higher inhibitory activity.
表1 細菌に対する各サンプルの阻止円の直径
表2 細菌に対する各サンプルのMIC値
Claims (4)
粉砕して篩にかけることにより得られた粉末を、まず35±5℃で、相対湿度60~70%の条件下で5~7日間置くことと、
粉末とpHが7.5~8.5である水とを固液質量比1:10~12で混合して2~3h静置して浸漬して、220~300℃で5~7h水蒸気蒸留することと、
を含むことを特徴とする、リステリア・モノサイトゲネスを阻害するヤクチ精油の抽出方法。 grinding and sieving the dried mature fruit of Yakuchi to obtain a powder;
The powder obtained by crushing and sieving is first stored at 35±5° C. and a relative humidity of 60-70% for 5-7 days ;
The powder is mixed with water having a pH of 7.5 to 8.5 in a solid-liquid mass ratio of 1:10 to 12, and left to stand for 2 to 3 hours for immersion, followed by steam distillation at 220 to 300 ° C. for 5 to 7 hours;
A method for extracting Yakuchi essential oil that inhibits Listeria monocytogenes, comprising:
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